Methods and devices for detecting a pathogen and its molecular components

ABSTRACT

Methods, systems and devices for detecting the presence of a pathogen, for example, a virus (e.g., SARS-CoV-2), or its molecular components, in health care-related samples and/or environmental samples are disclosed. An example system for improving detection of a pathogen includes biosensor device comprising a detection chip and at least one probe that specifically recognizes a pathogen, where the detection chip comprises a graphene field-effect transistor (FET) chip and the probe, which comprises an aptamer, specifically binds to a DNA, RNA, or protein associated with the pathogen.

CROSS-REFERENCE TO RELATED APPLICATION

This patent document claims priority to and benefits of U.S. ProvisionalApplication No. 63/144,460, entitled “METHOD AND DEVICE FOR DETECTION OFA VIRUS AND ITS MOLECULAR COMPONENTS,” and filed on Feb. 1, 2021. Theentire content of the before-mentioned patent application isincorporated by reference as part of the disclosure of this patentdocument.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. HL119893awarded by the National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“009062-8446.US01_ST25.txt” created on Apr. 1, 2022 and is 2,176 bytesin size. The sequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

TECHNICAL FIELD

This patent document generally relates to detection of pathogens, andmore specifically, to using field-effect transistors and aptamers toaccurately detect a virus and its molecular components.

BACKGROUND

Pathogens such as viruses and bacteria pose a significant concern forhuman health. For example, coronaviruses are a large family of RNAviruses that may cause upper-respiratory tract diseases in humans thatrange from mild to lethal in severity. The most recent novel coronavirusto cause worldwide pandemic and health care crises is SARS-CoV-2, whichcauses coronavirus disease 2019 (COVID-19). SARS-CoV-2 emerged inDecember 2019 and was declared a global pandemic by the World HealthOrganization (WHO) on Mar. 11, 2020. According to recent report,COVID-19 is highly contagious (>96 million positive cases) and causes ahigh morbidity rate (>2 million death) worldwide.

To successfully combat the pandemic, coordinated implementation ofmultiple population-scale health care measures is required. One of thecornerstones of this multi-probe strategy is everyday real-time accessto reliable, fast, inexpensive at-home, workplaces, and point-of-care(POC) diagnostic tests which would be carried out on simple in usediagnostic devices with wireless data transmission capabilities forcontinuing pandemic monitoring. Despite tremendous effort in this area,no such methodologies and devices exist.

SUMMARY

The disclosed technology relates to methods, systems, and devices fordetecting the presence of a pathogen, for example, a virus (e.g.,SARS-CoV-2), or its molecular components in health care-related samplesand/or environmental samples.

In some example aspects, a biosensor device is provided to detect thepresence of at least a finite count/amount of a viral particle, RNA,DNA, or protein associated with a pathogen of interest, for example, theSARS-CoV-2 virus. However, the specificity of the biosensor can beadvantageously changed to detect biological pathogens other thanviruses, e.g., flu, bacteria, toxins, or fungi. Thus, embodiments of thedisclosed technology can be deployed in efforts that range from globalheath to global security.

In other example aspects, the biosensor device comprises a detectionchip, for example, a graphene field-effect transistor (FET) chip. Insome embodiments, the detection chip comprises a probe attached to thedetection chip, for example, an aptamer, that specifically binds to atarget the viral antigen, particle, RNA, DNA, or protein. In someembodiments, the aptamer is an oligonucleotide (RNA or DNA,single-stranded or double-stranded). In some embodiments, the aptamer isa peptide.

In yet other example aspects, methods are provided for detecting whethera subject has been exposed to a particular virus, for example, theSARS-CoV-2 virus, by using the biosensor device of the presenttechnology to detect the presence of the viral particle, RNA, DNAs, orprotein associated with the virus in a sample from the subject.

In yet other example aspects, devices biosensor devices are provided fordetecting one or more pathogens. In an example, the biosensor deviceincludes a detection chip, which includes (a) a substrate with agraphene surface, (b) a conducting material at a first end and a secondend of the graphene surface that form a first electrode and a secondelectrode, respectively, and (c) an insulating material to insulate thefirst electrode and the second electrode. In this example, one or moreprobes, which are attached to the graphene surface, specifically bind toone or more target molecules of the one or more pathogens. Furthermore,the insulating material forms a well to receive a biological sample suchthat the biological sample is in contact with the one or more probes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C and 2A-2C are schematics showing the design and mechanism ofa portable chip-based electronic biosensor device with a wirelesscommunication module that facilitates efficient detection of viralRNA/DNA targets and surveillance of the viral pandemic, in accordancewith the presently disclosed technology.

FIG. 3 is a schematic showing the collection of the biological sampleand the detection of the pathogen using a handheld device.

FIG. 4A shows an example of a graphene FET (GFET) sensor on a chipcarrier and a breadboard setup for graphene FET sensors.

FIG. 4B shows an example drain-source current analysis with respect togate voltage for the experimental setup shown in FIG. 4A.

FIGS. 5A-5F show stages in an example fabrication of the graphene FET.

FIG. 6 shows a two-dimensional view of an example graphene FET.

FIGS. 7A-7F show various stages of atomic force microscopic imaging ofan example graphene FET.

FIGS. 8-11 show partial cross-sectional views of example embodiments ofa detection device in an assembled state.

FIG. 12 shows another example embodiment of a detection device.

FIGS. 13A and 13B show examples of assembled portable, compact devices.

FIG. 14-17 show examples of detection of SARS-CoV-2 using aptamersspecific to viral proteins with high specificity and sensitivity.

FIGS. 18A-18C shows an example Aptamer-S and Aptamer-N derivatized GFETsensor response due to binding of different concentration of RBD regionand N-protein of SARS-CoV-2.

FIGS. 19A-19F show example results for sensor specificity based ontesting aptamer-S and -N by using closely correlated cognate antigens ofMERS-CoV, SARS-CoV and inactive MERS-CoV virus.

FIGS. 20A and 20B show examples results for a concentration dependentsensor response with different concentrations of inactive viruses and10% saliva for the Aptamer-S derivatized GFET and the Aptamer-Nderivatized GFET, respectively.

FIG. 20C shows results of oral swab samples of RT-PCR positive andnegative tests corresponding to point-of-care (POC) antigen tests fromthe FDA EUA.

FIG. 20D shows results of a blind sample test using the Aptamer-Sderivatized graphene FET.

FIGS. 21A and 21B show examples of the mediated transfer curve analysisof the aptamer derivatized graphene FET.

FIGS. 22A-22C show examples of detecting the SARS-CoV-2 RBD cognateprotein of mutant viruses.

FIG. 22D shows an example of detecting the SARS-CoV-2 RBD cognateprotein of the original virus.

FIGS. 23A and 23B show example results from analyzing the specificity ofthe aptamer derivatized graphene FET.

FIGS. 24A-24G show example results for the current-to-voltage (I-V)relationship of aptamer-anylate interactions.

FIGS. 25A-25C shows example GFET device responses to the Delta variantN-protein, the Omicron variant N-protein, and the inactivated Deltavariant, respectively.

FIGS. 26A and 26B show example detection results of human saliva samplesduring the January 2022 Omicron wave using embodiments of the disclosedtechnology.

DETAILED DESCRIPTION

Disclosed are methods, devices and systems that pertain to the detectionof proteins, nucleic acids, and viruses and/or other pathogens such asbacteria, fungi, or biological toxins. In particular, disclosed are aportable wireless electronic biosensor device comprising a biosensorchip for the detection of a pathogen (e.g., a virus like SARS-CoV-2) orits molecular components from a biological sample, e.g., a sample ofbodily fluid (e.g., saliva, sweat, tears, exhaled breath, blood, urine)from a person, nasal, nasopharyngeal, or fecal swabs of a person, or inenvironmental samples, e.g., air, water, air borne pathogens, andhousehold and industrial waste. The hand-held, self-contained, portable,diagnostic device incorporates wireless communication technology andallows highly specific and sensitive pathogen detection with resultsreported within minutes. The described embodiments advantageously enablerapid and precise identification of SARS-CoV-2 and potentially otheremerging viral antigens around the world.

Example Embodiments in Global Health Applications

A person infected with a pathogen, for example the SARS-CoV-2 virus, mayeject the virus or tiny aerosolized droplets containing the virus bycoughing, speaking, or otherwise exhaling breath. A person's saliva mayalso contain measurable levels of the virus. A device comprising adetection chip indicating the presence of a target protein or nucleicacid associated with a particular virus, for example the SARS-CoV-2virus, could therefore be breathed onto or into by a user, or a dropletof saliva from the user could be deposited into the device to test theuser for the presence of the SARS-CoV-2 virus. Such a device capable ofsignaling the presence of the SARS-CoV-2 virus would benefit from beingportable and reusable for the testing of multiple persons. However, areusable device would necessitate a careful distinction between reusableand disposable portions of the device such as, for example a mouthpiecedisposed to contact the mouth of a user, to prevent inadvertentinfection of uninfected users via using a contaminated device.

Amidst a global pandemic associated with SARS-CoV-2 virus, and the riskof similar emerging outbreaks, or even mutations thereof, there exists aneed to quickly, safely, and easily test people for the likely presenceor absence of the SARS-CoV-2 virus, or other viruses related to disease.It would be beneficial to be able to test people to assess the presenceor absence of a disease related virus in their breath or saliva withminimal delay and risk of transmission between persons. For example, itwould be beneficial to quickly, safely, and easily test people within agiven population sample or location that may include for example withoutlimitation, an airport, a library, a theater, a classroom, a restaurantor bar, an office setting or lobby, a hotel or inn lobby/intake, placesof public transportation, hospital/urgent care/doctor's offices, or anyother internal space where people may gather and where transmission ofthe virus between persons is possible.

Furthermore, the portability of the described embodiments would enablethe biosensor devices to be deployed in areas that may be ascertained assources of the pathogens, e.g., caves in the example of SARS-CoV-2. Thisefficacy of the deployment is bolstered by the wireless connectivity andcapabilities of the biosensor device, which would be able toperiodically update research and medical facilities, and the sensitivityof the graphene FET, which would be able to detect very lowconcentrations of the pathogen, thereby enabling early detection at thesource.

Example Embodiments in Global Security Applications

Biological threats. Biological threats can emerge without warning fromnature, deliberate attacks, or accidental release. Infectious diseasesthat sweep the world, e.g., MERS-CoV and SARS-CoV-2 as discussed above,pathogens that are accidentally released from research laboratories,including from laboratories working on non-circulating viruses, e.g.,smallpox, or from research work that has created novel epidemic strainsof pathogens, can only be brought under control after enormousinternational collaboration with governments in the region that havebeen affected and many billions of dollars spent. Inter-connected traveland commerce, especially from regions in which the pathogens haveoriginated, quickly result in the spread of a pathogen all over theworld.

Governments have frequently relied on the private sector to make thevaccines, medicines, diagnostics and medical equipment etc. that areneeded to respond upon the emergence of biological threats. Embodimentsof the disclosed technology would be critical in the establishment ofnational and sub-national monitoring systems that can predict andidentify infectious disease threats. The sensitivity of the grapheneFET, which enables detection of very low concentrations, and theportability and cost-effectiveness of the described biosensor devicescan be leveraged in deployments that can serve as large-scale earlywarning systems for biological threats.

Environmental monitoring. The ability to deploy the described biosensordevices on a large-scale make them well suited for environmentalmonitoring, and in particular, for the detection of environmentalcontaminants such as heavy metals, small-molecule agricultural toxins,and water-borne bacterial pathogens. Additional targets include aquatictoxins, pesticides, industrial byproducts, antibiotics, andpharmaceuticals. Traditional multi-step detection processes may degrademany environmental contaminants of interest, which may already be at lowconcentrations. Embodiments of the disclosed technology provide on-sitedetection and the dissemination of results via built-in wirelesscapabilities, which make the described aptamer-based biosensorsparticularly useful for monitoring water, soil, and air.

Advantages and Benefits of the Disclosed Technology

Therefore, there exists a need for a device that can test for aplurality of disease-related pathogens via a single test. It would bebeneficial for such a device to be hand-held, self-contained, andportable, with a known test result generated within minutes of testing.It would be further beneficial for such tests to be cost-effective andthe results to be immediately transmitted to non-technical personnel aswell as doctors, administrators, and public health individuals. Such adevice would further benefit from the ability to test positive forvariants of pathogens, which allow for diagnosing potential futuremutations of known virus genomes, for example the SARS-CoV-2 genome,using aptamers or nucleic acids against such variants.

Embodiments of the disclosed technology employ graphene FET-biosensorchips, which have been used in conjunction with double-stranded probesfor detecting nucleotide acids with a single nucleotide resolution viastrand displacement (e.g., see U.S. Pat. No. 10,793,898 that disclosesgraphene field-effect transistor (FET)-based electrical biosensorchips). Compared with optical sensors, electrical biosensors have higher(Atto-Pico molar) specificity and thus reduces the need for sampleamplification. FET-based sensors, in particular, can sense change inelectric charges during biomolecular interactions and affords highestpossible sensitivity as every atom on the surface is used, i.e., singleelectron charge sensitivity. Electrical biosensors also allow fasterread-outs, low power consumption, portability, inexpensive massproduction, integrated sensor and measurement systems, and no labelingof biomolecules.

However, compared with known double-stranded nucleic acid probe andchip-based devices, the present technology has extended and superiorcapabilities, including the use of single stranded aptamers as probes,the detection of the electrical current resulting from simpleprobe-sample interactions, and the ability to design aptamers specificto nucleic acid targets as well as proteins of interest (e.g., spikeproteins of SARS-CoV-2). These features allow for versatility in theapplication of aptamers for nucleic acids, protein, and other sensingneeds, making the current technology simpler and tunable.

Embodiments of the disclosed technology provide, amongst other featuresand benefits, the following distinct advantages:

(1) Aptamers, selected from amongst thousands of available aptamers,that are able to bind to specific proteins;

(2) Aptamers that retain their specificity even when attached to asolid; and

(3) Aptamers that can recognize mutations of a pathogen.

Publicly available aptamer databases, e.g., Apta-index by Aptagen,contain sequences drawn from hundreds of published experiments. Entriesin the database typically provide detailed, structured information aboutthe experimental conditions under which aptamers were selected and theirbinding affinity quantified. A variety of analytical techniques havebeen employed to identify the best candidates that bind to the specificproteins considered herein, and embodiments of the disclosed technologyuse these aptamers to detect, with high sensitivity and specificity,various pathogens.

Existing implementations that use aptamers have always used aptamers insolution to detect pathogens. A common limitation of aptamer-basedassays has always been the possible drastic changes in their bindingproperties due to their immobilization on solid substrates. However, thedescribed embodiments attach the aptamers to graphene surfaces with theaptamers unexpectedly retaining their specificity, and can thus beincorporated into an electrical/electronic detection framework with highsensitivity. Prior art systems that used aptamers in solution could notteach or suggest immobilizing aptamers on a solid graphene surface asdescribed in disclosed technology.

The described embodiments use aptamers (or equivalent, DNA), instead ofantibodies, to bind to specific proteins. Thus, the inherentdisadvantage of antibodies in detecting mutations is circumvented byusing aptamers. As is evidenced from the results presented in thispatent document, the SARS-CoV-2 virus, as well as its Omicron and Deltavariants, are detected accurately using the aptamer-based GFET devicesthat exhibit high specificity and high sensitivity.

While the present disclosure is capable of being embodied in variousforms, the description below of several embodiments is made with theunderstanding that the present disclosure is to be considered as anexemplification of the invention and is not intended to limit theinvention to the specific embodiments illustrated.

Section headings are used in the present document to improve readabilityof the description and do not in any way limit the discussion or theembodiments (and/or implementations) to the respective sections only.

Examples of Detection Chips

In some embodiments, the biosensor device of the present technologycomprises a detection chip. In some embodiments, the detection chip hassimilar structural and functional characteristics as those disclosed inthe '898 patent. For example, the detection chip can include a wafer orsubstrate made from silicon oxide. The substrate can be coated with agraphene surface. Electrodes are provided on the graphene layer, and asolution reservoir is created by an insulating the electrodes with, forexample without limitation, silicone rubber. In some embodiments, probes(e.g., aptamers) are brought into contact with the graphene layer fordetection of particular targets of interest.

In some embodiments, a current to voltage (I-V) curve of the detectionchip can be generated in the absence or presence of target molecules.Without being held to any particular theory, attachment of probes (e.g.,aptamers) to the graphene layer increases the resistance of the graphenelayer, which results in a baseline I-V curve for the detection chip inthe absence of any target molecules. Hybridization of the probe with thetarget molecule may generate a change in the affinity between the probeand the graphene layer, thereby resulting in changes in the resistanceof the graphene layer, detectable by a shift of the I-V curve comparedto the baseline curve. The more the probes hybridize or if more targetmolecules hybridize, the larger the change in resistance, and thegreater the shift in the I-V curve, which provides a way to determinenot only the presence of the targeted molecule but also a detectedconcentration of the target molecule.

Examples of Aptamer Probes

In some embodiments, the detection chip of the biosensor devicecomprises a probe specific for a target molecule associated with apathogen of interest for detection of the presence of the pathogen. Insome embodiments, the probe comprises an aptamer for specificrecognition of target molecules, for example, DNAs, RNAs, or proteinsassociated with a pathogen of interest, for example, a virus (e.g., theSARS-CoV-2 coronavirus). Aptamers are often oligonucleotide or peptidemolecules that are designed and generated to specifically bind totargets of interest, such as proteins or nucleic acids. Aptamers can bemade from nucleic acids (RNA or DNA) or peptides. In some embodiments,the aptamer comprises nucleic acid and is single-stranded. In otherembodiments, the aptamer comprises nucleic acid and is double-stranded.

In some embodiments, the detection chip may be configured with aparticular aptamer to detect the presence of a specific target DNA, RNA,or protein, and the aptamer may be either natively (e.g., manually) orsynthetically (e.g., using an automated high-throughput depositionsystem) attached to the detection chip. In some embodiments, thedetection chip can include multiple aptamers each targeting a particularDNA, RNA, or protein of interest. In some embodiments, the multipleaptamers can be arranged in an array for testing the presence of one ora plurality of target DNAs, RNAs, or proteins associated with one ormultiple pathogens of interest.

In some embodiments, the aptamer-based detection chip can detect thepresence of a target molecule (e.g., protein, nucleic acid, virus) by achange in the light refracted, reflected, fluoresced, absorbed and/oremitted by the detection chip. Alternatively, the aptamer-baseddetection chip can utilize changes in impedance, resistance,capacitance, voltage, current, resistance, any combination thereof, orchanges in the electric field to indicate the presence of the targetmolecule bound by the aptamer.

As demonstrated in the working example, the biosensor device of thepresent technology can be used for rapid and precise detection of apathogen of interest, for example, the SARS-CoV-2 virus. However, thebiosensor device can be designed for any other pathogen of interest orconstructed to simultaneously detect multiple viruses, for example,common influenza, MERS-CoV, SARS-CoV, and SARS-CoV-2, by modifying theaptamer probes specific for the intended targets. The aptamers can alsobe adapted for detection of potential future variants of a targetpathogen, for example, potential future mutations within SARS-CoV-2genome.

Examples of Biosensor Devices

In some aspects, a biosensor device is provided for determining thepresence of a protein or viral particles in health care-related settingsand/or environmental samples (e.g., as shown in FIGS. 1A-1C and 2A-2C),with the collection of the biological sample shown in FIG. 3. In theexample shown in FIG. 3, the biological sample containing the protein orviral particle may be collected using a nasal swab, a pharyngeal swab,or saliva. In other examples, exhaled breath or environmental samples(e.g., air, water, household and industrial waste) may be collected.FIG. 3 further shows the sensor response readout, which is validatedagainst cognate proteins and virus samples, may be collected on ahandheld device.

In some embodiments, the biosensor device comprises a housing unithaving a first opening, a second opening, and a third opening. Acartridge is adapted to be removably fixed within the housing tofacilitate the measurements. The cartridge comprises a detection chipthat is in electrical communication with a surface of the cartridgeduring detection of a target. A cap is removably affixed over the secondopening, and the cap includes circuitry and a visual indicator. When thecartridge is disposed within the housing with the cap disposed over thesecond opening, the target-sensing detection chip is disposed in fluidcommunication with the first opening and the cap is disposed inelectrical communication with the surface of the cartridge.

In some embodiments, the detection chip (which can be mounted on abreadboard as shown in FIG. 4A) utilizes electron mapping or electrondensity mapping to distinguish a change in energy between a singlenucleotide pair. Such identification of the change in energy determinesthe proteins that comprise the nucleotide pair. The example setup shownin FIG. 4A results in the drain-source current analysis with respect togate voltage, as shown in FIG. 4B. In this example, the gate voltage wasscanned in the range of +1 V to −1 V with a step size of 2 mV, thedrain-source voltage (Vas) was 30 mV (which was optimized in the 0-100mV range in increments of 10 mV), the drain-source current (las) was onthe order of μA, and the Dirac voltage was analyzed at the I_(ds)minima.

In some embodiments, the detection chip, upon detection of one or moretargets, for example, viruses, can transmit that information in any of anumber of ways. In some embodiments, the detection chip can be connectedvia circuitry to one or more colored lights, for example LEDs, andsignals the illumination of a different color or colors preselected torepresent a detection event. The color or colors, or the intensitythereof, or the number of individual LEDs illuminated could also be anindication of the concentration of the detection event. In otherembodiments, the detection chip can be electrically connected withcircuitry that includes a wireless transmitter that transmits dataregarding the detection event to a computer or tablet or other portableor non-portable data storage device for analysis and/or later display.

FIGS. 5A-5F illustrate the stages in an example fabrication of thegraphene FET. As shown therein, the source drain electrodes (Au/Cr˜100nm) are deposited on an SiO₂ substrate (FIG. 5A) using sputteringdeposition, which is followed by depositing a passivation layer (SiO₂ orAl₂O₃˜80 nm) on the source drain electrodes (FIG. 5B). In FIG. 5C, thegraphene is wet transferred onto the patterned substrate and thePoly(methyl methacrylate) (PMMA) is removed by dissolving with acetone.In the next step, as shown in FIG. 5D, the (polymer, polymethylglutarimide) (PMGI) photoresist is applied to protect the sensor area,and the extra graphene layer is removed by O₂ plasma etching. The PGMIphotoresist is lifted off and the graphene FET is annealed in forminggas, e.g., a hydrogen/nitrogen atmosphere (FIG. 5E), and finallypolydimethylsiloxane (PDMS) or epoxy is applied to form a well forcontaining the sample liquid.

In some embodiments, the graphene FET may be configured to detectmultiple distinct pathogens by attaching multiple probes (e.g.,aptamers) on non-overlapping portions of the graphene FET. Each of themultiple probes attached is selected to bind to different specificproteins. In other embodiments, an array of detection chips can be usedto detect multiple distinct pathogens. In this example, each of themultiple probes is attached to a corresponding one of the array ofdetection chips. Thus, embodiments of the disclosed technology providealternate ways of detecting multiple pathogens.

FIG. 6 illustrates a two-dimensional schematic of the graphene FET,wherein the source, drain and gate electrodes are fabricated using goldpads, upon which there are passivating layers, and then the graphenelayer. In an example, the graphene FET includes a 500 μm channel inbetween the passivating layers adjacent to the gate and the drain sourceelectrodes.

FIGS. 7A-7F illustrate atomic force microscope imaging of the grapheneFET after different steps. FIG. 7A shows the bare graphene surface (witha roughness of 0.7±0.3 nm) and FIG. 7B shows the graphene surface aftera 5 mM PBASE addition (with a roughness of 1.8±0.5 nm).

FIG. 7C shows the Raman spectroscopy of pristine graphene and PBASEfunctionalized graphene FET (with the inset showing the light microscopeimage of the scanned area). The graphene FET was derivatized using PBASE(5 mM) and was analyzed in variable forward (−1 to +1 V) and reverse (+1to ˜1 V) gate voltage swipe of 0.2 V step and variable drain sourcevoltage (Vas) of 0-100 mV with incremental step of 10 mV. In addition tothe expected G- and 2D-peaks (with a peak intensity ratio of >1:2,indicating high quality material) on the pristine/untreated sample, theappearance of D and D′ peak with the addition of the PBASE might be dueto pyrene group binding, and enhanced spa bonding.

FIGS. 7D and 7E show the drain-source at 0 to −1 V, wherein the I_(ds)of the graphene FET is measured with respect to V_(G) and V_(SD),respectively. FIG. 7F shows the V_(D) hysteresis analysis at differentV_(ds) when cyclic (back- and forth-) gate voltage sweep in the range of+1 V to −1 V. As evidenced in FIG. 7F, in the range of 20 mV>V_(SD)>50mV there is a minimal hysteresis in the V_(D).

In some embodiments, a schematic representation of a biosensor device100 according to the present technology is illustrated in partialcross-section in an assembled state in FIG. 8 and shown in partialcross-section with the major components exploded in FIG. 9. As showntherein, the major components include a housing 110 that can, forexample, serve as a handle and a chassis for supporting the other majorcomponents. In some embodiments, the housing 110 and the majorcomponents are arranged along a longitudinal centerline 105. In otherembodiments, the components can be arranged in any geometry asaesthetically or functionally may be desirable, for example asillustrated in FIGS. 10 and 11 described further hereinbelow.

Referring to FIGS. 8 and 9, in one embodiment the housing 110 includes afirst opening 120, a second opening 130 and at least one third opening140. An insert or cartridge 150 is adapted to be removably fixed withinthe housing 110. For example, the cartridge 150 in one embodiment couldbe removably fixed by a threaded connection through the second opening130. In another embodiment the cartridge 150 includes a shoulder 160that extends laterally from an end of the cartridge 150 so that when thecartridge 150 is disposed within the housing 110, the shoulder 160overhangs an edge of the second opening 130 and is compressively heldagainst the edge of the second opening 130 by a cap 170 that attaches,for example by threads, over the shoulder 160 of the cartridge 150 atthe second opening 130. In other embodiments the cartridge 150 could beremovably fixed within the housing 110 by either of the above disclosedmechanisms and/or by a press fit or a magnetic attachment or any singleattachment mechanism or combination of attachment mechanisms as known inthe art.

Referring to FIGS. 10 and 11, in another embodiment of a device 200, ahousing 210 includes a first opening 220, a second opening 230 and atleast one third opening 240. In this embodiment an insert or cartridge250 is adapted to be removably fixed within the housing 250. Forexample, the cartridge 250 in one embodiment could be removably fixed bya threaded connection through the second opening 230. In anotherembodiment the cartridge 250 includes a shoulder 260 that extendslaterally from an end of the cartridge 250 so that when the cartridge250 is disposed within the housing 210, the shoulder 260 overhangs anedge of the second opening 230 and is compressively held against theedge of the second opening 230 by a cap 270 that attaches, for exampleby threads, over the shoulder 260 of the cartridge 250 at the secondopening 230. In other embodiments the cartridge 250 could be removablyfixed within the housing 210 by either of the above disclosed mechanismsand/or by a press fit or a magnetic attachment or any single attachmentmechanism or combination thereof as known in the art.

Regardless of the geometry of the housing 110, 210 in regard to how themajor components fit together, whether as shown in FIGS. 8-11 or usingother geometries as are known in the art for a housing with aninsertable and removable insert or cartridge, all embodiments of thecartridge 150, 250 include a detection chip 300 that is in electricalcommunication with a surface 310 of the cartridge 150, 250.

In some embodiments the detection chip 300 is reusable through acleansing process so that the cartridge 150, 250 on which it is disposedis also reusable. In other embodiments the detection chip 300 is asingle use chip so that the cartridge 150, 250 is a disposable cartridge150, 250. The detection chip 300 is electrically communicative to thesurface 310 for example, wirelessly, via wires 320, or traces or aninternal circuit board having wires or traces.

Still referring to FIGS. 8-11, in some embodiments a cap 170, 270attaches, for example by threads, over the second opening 130, 230 sothat circuitry 330 within the cap 170, 270 is in electricalcommunication with the surface 310, and therefore also in electricalcommunication with the detection chip 300. The cap 170, 270 in otherembodiments attaches over the second opening 130, 230 by a press fit, asnap fit, a magnetic attachment, a latch mechanism or by any othermechanism for removable attachment as may be known in the art.

The circuitry 330 is of the type as known in the art that can interfacewith a signal from the detection chip 300 and relay or send anindependent signal to a visual indicator 340 disposed on an outside ofthe cap 170, 270. The visual indicator 340 in one embodiment is one ormore LEDs but in other embodiments can be one or more incandescentbulbs, an LED or LCD digital display, or other sorts of visualindicators as may be known in the art. Like the description hereinabovefor the detection chip 30, the visual indicator 340 signals theillumination of a different color or colors preselected to represent adetection event. The color or colors, or the intensity thereof, or thenumber of individual LEDs illuminated could also be an indication of theconcentration of the detection event. In another embodiment the visualindicator 340 is electrically connected with the circuitry 330 thatincludes a wireless transmitter that transmits data regarding thedetection event to a computer or tablet or other portable ornon-portable data storage device for analysis and/or later display. Asillustrated in Figures FIGS. 8 and 10, when the cartridge 150, 250 isremovably fixed within the housing 110, 210 with the cap 170, 270disposed over the second opening 130, 230, the detection chip 300 isdisposed in fluid communication with the first opening 120, 220.

Referring now to FIG. 12, in another embodiment of a device 400, ahousing 410 includes a port for insertion of a detection chip 420 havingall the structural and functional features of the detection chips. Inthis embodiment the housing 410 further includes circuitry 430, and avisual indicator 440, both of which function the same as the circuitry330 and visual indicator 340 described hereinabove. The detection chip420 when inserted into the housing 410 functions in the same way as thedetection chip 300 by having electrical connections on a side thatcommunicate electrically with the circuitry 430. In this embodiment, thedetection chip 420 can be exposed to sample molecules, for example, byapplying saliva to the chip 420 or by breathing or coughing onto thechip 420.

Referring to any of the embodiments in FIGS. 8-12, a power source 350,for example one or more batteries or a battery pack is schematicallyshown as disposed within the device 100, 200, 400 and is in electricalcommunication with the circuitry of that embodiment. For example, in theembodiments shown in FIGS. 8-11 the power source 350 is disposed withinthe cap 170, 270 and is in electrical communication with the circuitry330. Therefore, when the cap 170, 270 is installed on the housing 110,210, the power source 350 is also in electrical communication with thesurface 310, and therefore is further in electrical communication withthe detection chip 300. Therefore, the power source 350 can provideelectrical power not only to the internal circuitry 330 within the cap170, 270, but can also provide electrical power to the detection chip300 when the device 100, 200 is assembled.

Still referring to FIGS. 8-11, in some embodiments a disposablemouthpiece 360 is removably attachable over the first opening 120, 220.In some embodiments the disposable mouthpiece 360 attaches, for exampleby threads, over the first opening 120, 220, whereas in otherembodiments the disposable mouthpiece 360 attaches over the firstopening 120, 220 by a press fit, a snap fit, a magnetic attachment, alatch mechanism or by any other mechanism for removable attachment asmay be known in the art.

In some embodiments prior to operation a fresh unused cartridge 150, 250is removably inserted into a housing 110, 210 and the cap 170, 270 isaffixed to the housing 110, 210. When the device 100, 200 is soassembled, for example, as illustrated in FIGS. 8 and 10, the detectionchip 300 on the cartridge 150, 250 is in fluid communication with thefirst opening 120, 220.

As noted above, a person's breath can carry a virus or tiny aerosolizeddroplets containing a virus and/or molecular components of said virusand certain viruses can be identified by a target protein or proteinsthat make up the virus. The detection chip 300, 420 can indicate thepresence of the target molecule, protein or proteins associated with theparticular virus, for example the SARS-CoV-2 virus. Therefore, inoperation, a user to be tested for the particular virus breathes intomouthpiece 360 (or otherwise onto the detection chip 300, 420) to testthe user for the presence of the SARS-CoV-2 virus. If the targetmolecule, and therefore the particular virus, is detected the detectionchip 300, 420 in association with the attached circuitry 330, 430 sendsa signal to the visual indicator 340, 440 disposed on the outside of thecap 170, 270 or housing 410. The visual indicator 340, 440 signals theillumination of a different color or colors preselected to represent adetection event. The color or colors, or the intensity thereof, or thenumber of individual LEDs illuminated could also be an indication of theconcentration of the detection event. In another embodiment the visualindicator 340, 440 is electrically connected with the circuitry 330, 430that includes a wireless transmitter that transmits data regarding thedetection event to a computer or tablet or other portable ornon-portable data storage device for analysis and/or immediate ordelayed detection event display. In some embodiments the visualindicator 340, 440 can additionally flash and/or illuminate to signal amalfunction, low battery, or other error or problem.

Returning now to FIGS. 8-11, third openings 140, 240 allow for theuser's breath to exit the housing 110, 210 without causing a pressurebuildup therein. Making the mouthpiece 360 disposable allows for a freshmouthpiece 360 to be installed on the device 100, 200 prior to each usethus lowering the risk of contamination between those persons tested.

In other embodiments illustrated for example as portions of FIGS. 8 and10, the cartridge 150, 250 and cap 170, 270 can be attached to oneanother directly by any of the method of attachment as describedhereinabove or as otherwise known in the art and without the housing110, 210. In these embodiments a user need only breathe onto thedetection chip 300 to be tested for the presence of a target protein andtherefore the associated virus.

In any of these embodiments, the biosensor device can be embedded indifferent assemblies and products, or appended, affixed, or removablyaffixed to clothing or hats via a clip, hook and loop fastener, or otherfastening mechanisms known in the art that can accommodate a detectionchip of this invention and hook or append the same to a target surface,such as, for instance, a hat or a mask. Moreover, the detection chip canbe replaceable or disposable. Furthermore, the detection chip can beused to detect more than one virus' presence by including an aptamerspecifically created to detect the presence of each of a plurality ofdifferent virus' nucleic acid or protein with particularity, each beingidentifiable by having a different color or colors, or illuminationpattern coordinated with a detection event.

There are several unique features of the provided biosensor device. Forexample, first, the biosensor device possesses multi-target diagnosticcapabilities, including (i) detection of viral particles with aresolution of less than 7 particles/sample; (ii) detection of molecularcomponents of said virus, including viral proteins, with detection limitin low nanomolar range; and (iii) detection of nucleic acids with singlenucleotide resolution and femtomolar sensitivity. Second, the sensorsurface is specifically processed and tuned to be charge sensitive to ahigher degree of specificity. Third, electrical recording and electronicdata analysis algorithm are designed to increase S/N ratio todistinguish smallest change in the Dirac potential minima. This allowsfor recording interaction of sample (e.g., virus, spike proteins) toprobe (e.g., aptamer) at the highest resolution (lowest number) with lowpower consumption. These features allow miniaturization and portabilityof the device.

Because of the design features, the biosensor device can achieve thefollowing: (i) read-out in 10 minutes; (ii) sample can be from saliva,aerosols, and body fluids, e.g., nasal or nasopharyngeal; (iii) highaccuracy (˜95%); (iv) sensitivity to as few as 20-30 viruses whichenables early detection; (v) wireless contact tracing; (vi) portabilitywith low power (9V battery) requirement and cell phone-comparabledimension; (vii) inexpensive mass production ($10/test) capability; and(viii) non-technical operation requirement, i.e., easy-to-use withlayman's training without any medical professional help.

Examples of Using the Biosensor Device for Rapid Covid-19 Testing

In this example, a portable diagnostic device for highly specific andsensitive detection of coronavirus SARS-CoV-2 was developed (FIGS. 1A-1Cand 2A-2C). The device contains high affinity aptamers against the spikeproteins of SARS-CoV-2 for active virus screening and records anelectrical output to indicates a positive response. The device hasin-built wireless functionality which allows rapid tracing andcommunication with interested decision makers (e.g., doctors,administrators, policy makers). The final assembled portable and compactdevice, with electronics integrated with the sensor chip, is shown inFIGS. 13A and 13B. It includes a readily integrable genomic informationworkflow, using a microfluidic module to achieve genome-scale coveragewith limited pre-processing and will allow diagnosing potential futuremutations within SARS-CoV-2 genome, using aptamers against suchvariants. A patient sample can be tested using the multi array-sensorsand the positive/negative results will be transmitted wirelessly.

Although originally developed for RNA/DNA detection at a 10 XMsensitivity, the device has been adopted to derivatize DNA aptamers tothe 2D-transistor to recognize their cognate partner within themicrofluidic device (FIG. 2A, right). Electrical signals resulting fromthe interaction of the sample with their specific bait and prey arerecorded digitally and transmitted by the integrated wireless system(FIG. 2C). As designed, the device enables concentration and specificitydeterminations of antibodies, and detection and concentrations ofantigens.

In this example, the device focused on the receptor binding domain ofSARS-CoV-2 and specific fragments of the Spike protein, and DNA aptamersequences specific for these antigens were used. An electrical outputresponse indicates a positive response. As shown, the electricalgraphene FET (GFET) sensor can detect as low as 7 viral particles fromdiluted human saliva samples, without qPCR amplification, in 10 minutesusing aptamers specific to two different viral proteins: Aptamer Np-48(Aptamer 48) specific to the N-protein and Aptamer 1C specific to theS-protein (FIGS. 14-17), suggesting high specificity and sensitivity forSARS-CoV-2 testing. In particular, FIGS. 16 and 17 illustrate sensorresponse plotted on the y-axis against varying degrees of dilution ofSARS-CoV-2 virus-containing saliva samples plotted on the x-axis forAptamer Np-48 and Aptamer 1C, respectively. Individual data points arelabeled to indicate the number of the virus particles detected. Thedetection chip has demonstrated the ability to detect as few as 7 virusparticles.

Embodiments of the disclosed technology use aptamer derivatized GFETsfor label free detection and reporting of SARS-CoV-2 and its variant(N501Y, D614G, and Y453F) antigen, which enables the detection ofSARS-CoV-2 antigen, aptamer for S-protein (Aptamer 1C Kd≈5.8 nM)^([4])and N-protein (Aptamer Np-48 0.5 nM)^([5]) based on their affinity. Boththe aptamers were modified for GFET derivatization at 3′ end tofunctionalize the graphene surface. Further, both aptamers were analyzedfor the S-protein receptor binding domain (RBD) and N-protein usingcognate proteins, inactive virus, and retrospectively RT-PCR validatedoral samples.

Materials. The results presented herein were obtained by designing HPLCgrade 3′ amino functionalized aptamers for N-protein (Aptamer-N)^([5])and spike RBD (Aptamer-S)^([4]) to integrate with GFET. Molecularbiology grade 1×PBS (Gibco), MgCl₂, and Ultrapure water (Invitrogen)were used throughout the study. Analytical grade 1-pyrenebutyric acidN-hydroxyscuccinimide ester (PBASE) and ethanolamine were used withoutfurther processing.

Aptamer derivatization on GFET. The selected aptamers were aminoderivatized at 3′ end using linker molecule and labeled as Aptamer-S forspike RBD protein^([4]) and Aptamer-N^([5]) for Nucleocapsid protein.The aptamers were dissolved in 1×PBS buffer containing 0.5 mM MgCl₂ andannealing was performed by control heating at 94C for 2 min and slowcooling to room temperature. The annealed aptamer was stored at −20C forfurther usage. Derivatization was performed by adding 1 μM of aptamer onPBASE functionalized GFET for 30 min. Excess of aptamer was washed andunreacted PBASE were passivated using 10 mM ethanolamine (EA) solutionfor 20 min. The excess EA was washed and GFET measurement performed in1×PBS buffer.

Nucleocapsid and spike RBD domain detection. Baseline correction wasperformed using Aptamer functionalized GFET by sweeping the VG withinthe range of ±0.5 V while drain-source voltage maintained at fixedvoltage (100 mV). The concentration dependent sensor response wasanalyzed at different concentrations of cognate proteins (RBD (10, 20,50, 100, 200 nM))^([4]) and (Nucleocapsid ˜0.5, 1, 5, 10, 20, 50, 100nM)^([5]). After 10 min incubation, excess protein was washed threetimes using 1×PBS buffer and transfer function (ΔV_(D)) for sensor wasanalyzed. The sensor response was calibrated through a V_(D) shift perthe following relation:

ΔV _(D) =V _(D) −V _(D) ₀ .

Herein, V_(D) is the V_(D) after addition of sample on chip, V_(D) ₀ isthe V_(D) with aptamer derivatized chip, and the percentage response iscalculated with respect to V_(D)( ) (i.e., (ΔV_(D)/V_(D) ₀ )×100).Furthermore, the aptamer-GFET sensor to detect SARS-CoV-2 variants ofconcern such as B.1.1.7 (N501Y), Y453F and D614G[^(6-8]) using differentconcentration of recombinant RBD proteins. Two different concentration(100 fM, 100 nM) of mutant variant were utilized and the responsecompared with that obtained for the RBD of SARS-CoV-2.

Inactive virus detection in simulated conditions. The relatedexperiments were performed by preparing diluted solutions containingheat inactivated SARS-CoV-2 (USA-WA1/2020**, 9.55×10⁶ TCID₅₀/ml,Zeptometrix). The PFU/mL was calculated as described by Ding etal^([6]). The effect of the increment of virus dilution (6.68-6.68×10⁶PFU/mL) on GFET sensor response was measured. 10 μl sample was added onthe chip and incubated for 10 min and the V_(D) shift was measured inthe I_(ds)−V_(G) characteristics.

Specificity analysis. To analyze the specificity of the GFET sensor twodifferent concentrations of cognate proteins for MERS-CoV, SARS-CoV,SARS-CoV-2 were analyzed. Sensor response at an ultra-low concentration(100 fM) as well as in the saturation ranges (100 nM) using aptamers forRBD and N protein were investigated.

To analyze the sensitivity and specificity of different concentration ofcognate N and RBD proteins (100 fM-100 nM) of MERS-CoV, SARS-CoV,SARS-CoV-2 were used. Concentration dependent analysis was performed byusing different dilutions of inactive MERS-CoV and SARS-CoV-2 viruses(670 PFU/mL-6.7×10⁵ PFU/mL) in the simulated conditions described above.

Clinical sample analysis. The oral samples of patients were collected bytrained clinician in 3 ml of (0.9% w/v) saline approved by CDC andfurther tested by CLIA certified lab by trained clinician. RT-PCRanalysis was performed using FDA approved Promega RT-PCR test kit forSARS-CoV-2 while 10 μL of aliquot of same sample was used in the CLIAlab using GFET sensor and handheld reader. The Aptamer-S showed highersensor response with inactive virus in simulated environment and wasused for all the patient sample diagnosis. A total of thirty patientsamples were tested and compared retroactively with RT-qPCR (Ct value35) data. The known RT-qPCR negative data was used to set the sensorresponse threshold value with 99.7% of confidence interval (CI) using±3σ analysis to predict negative patient samples. The sensor responsevalue above the Mean+3σ was assigned as positive. The positive percentagreement (PPA) and negative percent agreement (NPA) of test werecalculated as described FDA guidelines^([10]).

The limit of detection (LoD) and limit of quantification (LoQ) of sensorwere estimated using standard deviation of the response and the slopemethod^([10]). All the data presented were the mean of at least threemeasurements and error of one standard deviation (SD).

Analysis of the SARS-CoV-2 and its mutant antigen. Due to wide range ofviral load in a patient sample (10⁴-10⁷ copy number/mL and 3.48 fM-58.9nM of antigen level)^([11][12],) the concentration dependent sensitiveregion as well as the saturation of sensor response were analyzed. Theanalysis of Aptamer-S and -N GFET sensor response in differentconcentrations of RBD and N protein of SARS-CoV-2 indicated aconcentration dependent exponential shift in the V_(D), as shown inFIGS. 18A-18C. As shown therein, the sensor reached saturation at 200 nMof RBD (FIG. 18A) and 100 nM of N-protein (FIG. 18B). It was observedthat all of the tested concentration of antigen 20% sensor response andafter saturation, the excess antigen level did not reduce the sensorresponse below 20% of response. This suggests that the GFET sensorreduces the chances of missing the positive signal due to hook effect asin the case of flow-based antigen testing^([13][14]).

The antigen test approved by FDA under emergency use authorization (EUA)(shown below in Table 1) did not indicate the ability to detect newvariant/s which may escape immunity generated by available vaccine orpast infection^([15]). There has also been considerable concern^([16])about false negatives of the recommended tests. The evolution of newmutations of SARS-CoV-2 (B.1.1.7 variant (N501Y), mink-related mutation(Y453F), mutation at S2 domain (D614G) are major matters ofconcern^([17]). Considering the importance of such issues, the GFETsensor was deployed on different SARS-CoV-2 mutants. It was indicated,through such tests, that Aptamer-S showed more than 20% of sensorresponse with 100 fM-100 nM concentration of respective proteins (asshown in FIG. 18C). Although, there is variant specific variability insensor response observed; however, sensor response was always above thethreshold value for positive sample (e.g., >20%) (see FIG. 18C). Basedon the concentration dependent analysis of cognate RBD and N protein onrespective aptamer derivatized GFET sensors with the SARS-CoV-2 variant,embodiments of the disclosed technology can detect relevant viralantigen at fM-nM concentration levels. A reason for the enhancedsensitivity, as well as the concomitant specificity, is due tonon-overlapping binding site of aptamer-S at spike protein amino acidT500, N437, and Q506^([4]) with new mutations.

TABLE 1 Comparison of point of care (POC) antigen test data collectedfrom the FDA Emergency Use Authorization (EUA) NPA % (n)/ Time EntityTarget/Readout LOD PPA % (n) (min) PIVOT- S/N proteins, S Aptamer-S 1.28100 (16)/ 15< Apta- Mutant proteins, PFU/mL and 100 (14) Sensor Droplet,Current- aptamer-N 1.45 (current) Voltage, PFU/mL BinaxNow N protein,140.6 98 (111)/ 15 Ag Card N.A., LF, TCID₅₀/ml 83.3 (50) Ellume Nprotein, 103.80 97 (148)/ 20 N.A., LF TCID₅₀/mL 95 (40) CareStart Nprotein, 8 × 10² TCID/ 99.32 (149)/ 10 LF, N.A. mL 93.75 (31) RepurposeS/N proteins, 6.31 [S]/5.27 100(8)/ 60 Glucometer aptamer, N.A., [N] pMprotein 100 (16) Glucometer Sofia 2 N protein, 113 TCID₅₀/ml 100 (179)/15 LF, FL, N.A. 96 (30) LumiraDx N protein, 32 TCID₅₀/ml 96.6 (174)/ 12FLI, N.A. 97.6(83)

Analysis of sensor specificity. The specificity of the disclosed sensorembodiments with aptamer-S and -N were tested by using closelycorrelated cognate antigens of MERS-CoV, SARS-CoV and inactive MERS-CoVvirus. As seen in FIGS. 19A and 19B, the results clearly indicate thataptamer-S and N significantly differentiate the MERS-CoV and the SARSvirus protein (>20% increment of sensor response). However, the grapheneFET was unable to significantly differentiate between SARS-CoV and CoV-2proteins. It was observed that Aptamer-N sensor response (50%) washigher with all tested protein samples compare to Aptamer-S (35%) andmay be ascribed to higher affinity of aptamer (K_(d), 0.5 nM compared toK_(d), 5.8 nM for Aptamer-S).

To further verify the specificity of aptamers, the scrambled aptamer-Sand Aptamer-N (as shown in Table 2 below) were used and derivatized thegraphene surface of the GFET. FIGS. 19C and 19D illustrate the relativeresponse of the aptamers (Aptamer-S, and -N) and the respectivescrambled aptamers (Aptamer-S, and -N) derivatized on GFET with inactiveSARS-CoV-2 virus in saliva diluted in PBS buffer. To further analyze thespecificity of the Aptamer-S and -N derivatized chip in simulatedbiological condition, different equivalent dilutions of inactiveSARS-CoV-2 and MERS-CoV in 10X v/v saliva in 1×PBS buffer were prepared.The concentration dependent analysis showed that all the dilutions ofinactive MERS-CoV samples showed less than 10% of sensor response (seeFIGS. 19E and 19F). One of the reasons for a high sensor response withSARS-CoV and SARS-CoV-2 may be the high affinity of aptamer-S at RBDamino acid position 500, 437, and 506 containing Threonine (T),asparagine (N) and glutamine (Q) while low sensor response with MERSmight be due to presence of different amino acid^([4][18]) whileMERS-CoV contain different amino acid (alanine (A), lysine (K), andalanine (A)) at same position of RBD.

TABLE 2  Nucleic acid sequences screened for embodiments of the disclosed technology SEQ ID Sequence (5′-3′) Ref. NOAptamer-S: ^([1]) 1 CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGC GTTAATGGACAAptamer-S1: ^([1]) 2 ATCCAGAGTGACGCAGCATTTCATCGGGTCCAAAAGGGGCTGCTCGGGATTGCGGATATGGACACGT Aptamer-N: ^([2]) 3GCTGGATGTCGCTTACGACAATATTCCTTAGGGGCACCGC TACATTGACACATCCAGC Aptamer-N1:^([2]) 4 GCTGGATGTCACCGGATTGTCGGACATCGGATTGTCTGAG TCATATGACACATCCAGCAptamer-N2: ^([3]) 5 GGGAGAGCGGAAGCGUGCUGGGCCUGUCGUUCGCUGUGUCUUGCUACGUUACGUUACACGGUUGGCAUAACCCAGAGGUC GAUGG Aptamer-N3: ^([3]) 6GGGAGAGCGGAAGCGUGCUGGGCCUCAUUACACACAUCUCACGGGAGACAUAGCUGACGAUAUCCAUAACCCAGAGGUCG AUGG Scrambled Aptamer-S: 7GGTGGTTTGACTGATCAGAGACTATGCAGCTCTGGCCCGC TAGCCGGAAGTScrambled Aptamer-N: 8 TAGGTATATGTACGCACACCTAAGTCAGCTTGCCCGACGCTGCAGCTCACATGTATGC

These results signify that both (Aptamer-S and -N) functionalizedsensors are specific for SARS-CoV-2 proteins in the virus. However, GFETsensor with aptamer-S showed higher sensor response compared toaptamer-N in simulated biological samples. Although earlier we haveobserved that it showed higher sensor response with cognate N-protein(see FIGS. 19A and 19B). Generally, the surface S-protein is more easilyaccessible compared to N-protein which is encapsulated within a lipidmembrane[¹⁹].

Limit of detection (LoD) and limit of quantification (LoQ). To analyzethe LoD and LoQ of our aptamer-based sensor, a concentration dependentsensor response analysis was performed with inactive viruses insimulated biological conditions. Triplicate data was fitted using linearfit and the LoD and LoQ calculated following FDA statistical dataanalysis guidelines. The results (shown in FIGS. 20A and 20B) indicate aLoD with aptamer-S of 1.28 PFU/mL (R²=0.98) and aptamer-N of 1.45 PFU/mL(R²=0.99) with inactive virus in 10% v/v saliva and 1×PBS buffer at roomtemperature. The estimated LoQ was 3.89 PFU/mL and 4.39 PFU/mL foraptamer-S and -N, respectively. The test showed higher sensitivity(lower LoD) compared to the FDA approved antigen test (see Table 1above). These results demonstrate the sensitivity of the aptamer basedGFET sensor to detect SARS-CoV-2 in biological fluids and provides themotivation for investigations with real patient samples.

Clinical sample collection and analysis. Based on the simulatedbiological sample analysis, it was observed that aptamer-S derivatizedGFET showed high sensor response while aptamer-N response was smaller(FIGS. 19D and 20B). Therefore, the aptamer-S derivatized GFET was usedfor patient sample analysis. To validate the GFET sensor with relatedsamples, single blind experiment was performed with 10 each negative andpositive sample and confirmed, independently through RT-PCR (as shown inFIGS. 20C and 20D). Moreover, 10 double blind samples were tested toconfirm the test. Based on the results, the RT-PCR negative sample wasused to predict with confidence interval (CI) of 99.7%+3a of sensorresponse, which yields 132.6 mV of the V_(D) shift (<20% of sensorresponse) for the samples. A threshold value of 132.6 mV was set with aV_(D) shift above this threshold being considered as positiveconfirmation of SARS-CoV-2 infection (see FIGS. 20C and 20D).

The efficacy of the disclosed technology is further evidenced in FIG.21A that shows an example of SARS-CoV-2 RBD binding mediated transfercurve analysis of Aptamer-S derivatized GFET, FIG. 21B that shows anexample of SARS-CoV-2 nucleocapsid protein binding mediated transfercurve analysis of Aptamer-N derivatized GFET, FIGS. 22A-22D that showexamples of detecting the SARS-CoV-2 RBD cognate protein of mutantN501Y, Y453F, D614G, and original virus, respectively, FIGS. 23A and 23Bthat show examples of analyzing the specificity of the Aptamer-S andAptamer-N derivatized GFET, respectively, and Tables 3-6 shown below.

TABLE 3 Negative Saliva Sample Test Results (SARS-CoV-2 virus clinicaltesting) Sample RTPCR PIVOT Biosensor Number Recorded Ct Mean DiracVoltage Reading N01 Negative >40 −10.30 N02 Negative >40 −7.20 N03Negative >40 21.23 N04 Negative >40 29.50 N05 Negative >40 31.28 N06Negative >40 35.05 N07 Negative >40 35.20 N08 Negative >40 46.20 N09Negative >40 89.70 N10 Negative >40 91.30 Mean PIVOT Biosensor DiracVoltage 36.20 Mean for negative samples Standard Deviation 33.89 99.7%(3x Standard Deviation) 101.68 Confidence Internal Upper Limit Baselinefor SARS-CoV-2 detection 137.88 (PIVOT Biosensor readings above thisvalue indicate SARS-CoV-2 virus present in sample)

TABLE 4 Positive Saliva Sample Test Results (SARS-CoV-2 virus clinicaltesting) PIVOT PIVOT Biosensor Biosensor Prediction Sample RTPCR DiracVoltage (>137.88 Number Recorded Ct Mean Reading Dirac voltage) P01Positive 18.37 217.19 Positive P02 Positive 24.35 205.80 Positive P03Positive 24.70 247.85 Positive P04 Positive 24.92 204.88 Positive P05Positive 26.19 136.10 Positive P06 Positive 28.08 532.68 Positive P07Positive 28.70 145.60 Positive P08 Positive 30.96 163.29 Positive P09Positive 32.14 149.55 Positive P10 Positive 32.60 226.60 Positive

TABLE 5 Blind Saliva Sample Test Results (SARS-CoV-2 virus clinicaltesting) PIVOT PIVOT Biosensor Biosensor Dirac Prediction Sample voltage(>137.88 RTPCR Number Reading Dirac voltage) Recorded Ct Mean B01 102.11Negative Negative >40 B02 83.20 Negative Negative >40 B03 68.80 NegativeNegative >40 B04 127.90 Negative Negative >40 B05 217.57 PositivePositive 20.92 B06 461.11 Positive Positive 22.80 B07 205.80 PositivePositive 24.35 B08 199.71 Positive Positive 27.46 B09 348.02 PositivePositive 29.63 B10 175.97 Positive Positive 27.67

TABLE 6 SARS-CoV-2-Delta/Omicron variant clinical test results PIVOTBiosensor PIVOT Sample Dirac Voltage Biosensor RT-PCR # Aptamer Readingprediction result Ct # 38 N 20.08 Negative Negative 0 (>40) 41 S 9.11Negative Negative 0 (>40) 51 S 1.36 Negative Negative 0 (>40) 59 N 8.80Negative Negative 0 (>40) 60 N 7.31 Negative Negative 0 (>40) 68 N 17.24Negative Negative 0 (>40) 44 S 110.18 Positive Positive 27.97 65 N136.31 Positive Positive 29.93 77 N 124.27 Positive Positive 27.58 43 S39.28 Positive Positive 25.48

Example results for the current-to-voltage (I-V) relationship ofaptamer-anylate interactions are shown in FIGS. 24A-24G. FIG. 24A showsthat, in the absence of the aptamer on the GFET, there is no change inthe Dirac potential shift, which motivates the need for the aptamer forsensing. The specificity of aptamer-omicron nucleocapsid protein bindingis shown in FIG. 24B, in which the Dirac potential is negligible when ascrambled (or control) aptamer is used. The significant shift in Diracpotential is seen in FIG. 24C, which shows the effect of a specificinteraction of aptamer-omicron nucleocapsid protein. The results inFIGS. 24A-24C are leveraged in the analyses of the results shown inFIGS. 24E-24G because currently there is no inactivated omicorn virusavailable.

FIG. 24D shows a dose response I-V plot of an aptamer for variousconcentrations of inactivated Delta variant virus. The results in FIG.24D are used to detect Delta variants in clinical samples, e.g., theexample results shown in FIGS. 24E-24G. As discussed in this document,the disclosed embodiments can detect as few as 5-10 viruses.

FIGS. 24E-24G show example results that include representative I-Vcurves for clinical testing in December 2021. As shown therein, in theabsence of the aptamer (sensor), there is no to little change in theDirac potential for both an individual whose RT-PCR tested positive(FIG. 24E) and an individual whose RT-PCR tested negative (FIG. 24F).However, for an RT-PCR positive individual (who is asymptomatic), thereis a significant shift in the Dirac potential when the aptamer ispresent (FIG. 24G).

FIGS. 25A-25C shows example GFET device responses to the Delta variantN-protein, the Omicron variant N-protein, and the inactivated Deltavariant virus, respectively. The results shown in FIGS. 25A and 25C wereobtained using a method of serial dilution to determine the doseresponse of the GFET system for detecting the Delta variant. The GFETsensors were functionalized using the N-aptamer. Nucleocapsid protein(N-protein) (2-4×10⁹ copy/ml of ORF1a) of the Delta variant SARS-CoV-2(B.1.617.2) were tested. The various dilutions were tested serially onthe PIVOT device using a single detection chip. The results shown inFIG. 25B were obtained using different detection chips, and each bar inFIG. 25C shows the shift from the baseline reading in saline to theviral omicron N-protein application of 10 μM. The control experimentsincluded detection chips that were not functionalized with any aptamers(the “No Aptamer” bar in FIG. 25C) or functionalized with a controlaptamer (e.g., a random sequence; the “scrambled N aptamer” bar in FIG.25C).

FIGS. 26A and 26B show example detection results of human saliva samplesduring the January 2022 Omicron wave for the N-aptamer and S-aptamer,respectively, using embodiments of the disclosed technology. As shown inFIG. 26A, a total of 15 samples were analyzed using N-aptamer GFETdevices, of which 12 samples resulted in a positive score (indicating anOmicron infection), yielding an estimated 80% infection rate in the US.Similarly, as shown in FIG. 26B, a total of 17 samples were analyzedusing S-aptamer GFET devices, of which 10 samples resulted in a positivescore (indicating an Omicron infection), yielding an estimated 59%infection rate in the US. In combination, 32 samples were analyzed usingeither N- or S-aptamer GFET devices, of which 22 samples resulted in apositive score, yielding an estimated 69% infection rate in the USpopulation.

Compared with available diagnostic devices on the market, the biosensordevice of the current technology is cheaper (˜$10/test), faster (˜10mins) and represents a portable point-of-care (POC) system withuniversal utility for current COVID-19 epidemic. The device also has thepotential to be adapted for fast and precise detection of othercoronaviruses (e.g., MERS-CoV, SARS-CoV), as well as mutants and/orvariants of the viruses with little modification. It requiresnon-technical operation and allows rapid transmission of results todoctors, administrators, and public health individuals, contributing tothe efforts in combating current and future pandemics.

Embodiments and Implementations of the Disclosed Technology

The disclosed technology includes devices biosensor devices fordetecting one or more pathogens. In an example, the biosensor deviceincludes a detection chip, which includes (a) a substrate with agraphene surface, (b) a conducting material at a first end and a secondend of the graphene surface that form a first electrode and a secondelectrode, respectively, and (c) an insulating material to insulate thefirst electrode and the second electrode. In this example, one or moreprobes, which are attached to the graphene surface, specifically bind toone or more target molecules of the one or more pathogens. Furthermore,the insulating material forms a well to receive a biological sample suchthat the biological sample is in contact with the one or more probes.The biosensor device is shown, for example, in FIGS. 1B, 2B, 3 and 6.

In some embodiments, at least one of the one or more probes is anaptamer.

In some embodiments, the aptamer comprises a nucleic acid or a peptide.In some examples, the nucleic acid aptamer is selected from Table 2. Inother examples, the nucleic acid is double-stranded. In yet otherexamples, the nucleic acid is single-stranded.

In some embodiments, the one or more pathogens are one or more variantsof a coronavirus. In some examples, the one or more variants of thecoronavirus include SARS-CoV, SARS-CoV-2, and MERS-CoV.

In some embodiments, the target molecule is a nucleic acid or a protein.

In some embodiments, the target molecule includes an S protein ofSARS-CoV-2, an N protein of SARS-CoV-2, a variant thereof, or a subunitthereof.

In some embodiments, the one or more probes comprise multiple probesthat are attached to different portions of the graphene surface. In someexamples, a single detection chip, e.g., as shown in FIG. 1B, 2B, 3 or6, can be configured to detect multiple pathogens using the multipleprobes that are attached to the graphene surface of the GFET of thedetection chip. This advantageously enables the described embodiments tobe used in environmental monitoring and in global security applications.

In some embodiments, a first probe of the multiple probes is attached toa first portion of the graphene surface, and a second probe of themultiple probes is attached to a second portion of the graphene surfacethat is non-overlapping with the first portion. In some examples, themultiple probes specifically bind to different target molecules of asame pathogen. In other examples, the multiple probes specifically bindto different target molecules of different pathogens.

In some embodiments, the biosensor device further includes a pluralityof detection chips comprising the detection chip, and each of themultiple probes is attached to a corresponding detection chip of theplurality of detection chips. In this embodiment, the describeddetection chips can be implemented as an array, which enables itsdeployment in environmental monitoring and global security applications,but also allows the plurality of detection chips to be processedsimultaneously.

In some embodiments, a handheld device, e.g., as shown in FIGS. 13A and13B, is configured to receive the biosensor device. In some examples,the handheld device is configured to perform a detection of the one ormore pathogens based on the one or more probes specifically binding tothe one or more target molecules of the one or more pathogens. In someexamples, the handheld device comprises a wireless transceiver that isconfigured to transmit a result of the detection. The wirelesstransceiver may support at least one of a Bluetooth protocol, a Wi-Fiprotocol, or a cellular protocol.

In some embodiments, the handheld device includes a power source, one ormore visual indicators, coupled to the power source, configured toindicate a start and a completion of the detection of the one or morepathogens, and a display, coupled to the power source, to present aresult of the detection for each of the one or more pathogens. In someexamples, the one or more visual indicators comprise LEDs, the displaycomprises an LCD, and the power source comprises one or more batteries.

Embodiments of the disclosed technology also provide a method ofdetecting the presence of the one or more pathogens in the biologicalsample obtained from a subject. The method includes contacting thebiological sample with the biosensor device described in any of theembodiments or implementations above.

In some embodiments, the biological sample comprises saliva, exhaledbreath, nasal swab, or nasopharyngeal swab of the subject.

In some embodiments, a presence of less than 10 particles of thepathogen in the biological sample is detected.

Embodiments of the disclosed technology support inter alia the followingtechnical solutions that solve the technical problem of accuratelydetecting one or more pathogens using a reliable, inexpensive, andportable device.

1. A biosensor device for detecting one or more pathogens, comprising adetection chip, comprising a substrate with a graphene surface, aconducting material at a first end and a second end of the graphenesurface that form a first electrode and a second electrode,respectively, and an insulating material to insulate the first electrodeand the second electrode, wherein one or more probes are attached to thegraphene surface, wherein the one or more probes specifically bind toone or more target molecules of the one or more pathogens, and whereinthe insulating material forms a well to receive a biological sample suchthat the biological sample is in contact with the one or more probes.

2. The biosensor of solution 1, wherein at least one of the one or moreprobes is an aptamer.

3. The biosensor of solution 2, wherein the aptamer comprises a nucleicacid or a peptide.

4. The biosensor of solution 3, wherein the nucleic acid isdouble-stranded.

5. The biosensor of solution 3, wherein the nucleic acid issingle-stranded.

6. The biosensor of any one of solutions 1 to 5, wherein the one or morepathogens are one or more variants of a coronavirus.

7. The biosensor of solution 6, wherein the one or more variants of thecoronavirus include SARS-CoV, SARS-CoV-2, and MERS-CoV.

8. The biosensor of any one of solutions 1 to 7, wherein the targetmolecule is a nucleic acid or a protein.

9. The biosensor of any one of solutions 1 to 8, wherein the targetmolecule includes an S protein of SARS-CoV-2, an N protein ofSARS-CoV-2, a variant thereof, or a subunit thereof.

10. The biosensor of any one of solutions 1 to 9, wherein the one ormore probes comprise multiple probes that are attached to differentportions of the graphene surface.

11. The biosensor of solution 10, wherein a first probe of the multipleprobes is attached to a first portion of the graphene surface, andwherein a second probe of the multiple probes is attached to a secondportion of the graphene surface that is non-overlapping with the firstportion.

12. The biosensor of solution 10 or 11, wherein the multiple probesspecifically bind to different target molecules of a same pathogen.

13. The biosensor of solution 10 or 11, wherein the multiple probesspecifically bind to different target molecules of different pathogens.

14. The biosensor of solution 10, comprising a plurality of detectionchips comprising the detection chip, wherein each of the multiple probesis attached to a corresponding detection chip of the plurality ofdetection chips.

15. The biosensor of any one of solutions 1 to 14, wherein a handhelddevice is configured to receive the biosensor device, and wherein thehandheld device is configured to perform a detection of the one or morepathogens based on the one or more probes specifically binding to theone or more target molecules of the one or more pathogens.

16. The biosensor of solution 15, wherein the handheld device comprisesa wireless transceiver that is configured to transmit a result of thedetection.

17. The biosensor of solution 16, wherein the wireless transceiversupports at least one of a Bluetooth protocol, a Wi-Fi protocol, or acellular protocol.

18. The biosensor of solution 15, wherein the handheld device comprisesa power source, one or more visual indicators, coupled to the powersource, configured to indicate a start and a completion of the detectionof the one or more pathogens, and a display, coupled to the powersource, to present a result of the detection for each of the one or morepathogens.

19. The biosensor of solution 18, wherein the one or more visualindicators comprise light-emitting diodes (LEDs), the display comprisesa liquid crystal display (LCD), and the power source comprises one ormore batteries.

20. A method of detecting the presence of the one or more pathogens in abiological sample obtained from a subject, the method comprisingcontacting the biological sample with the biosensor device of any one ofsolutions 1 to 19.

21. The method of solution 20, wherein the biological sample is selectedfrom the group consisting of saliva, exhaled breath, nasal swab, ornasopharyngeal swab of the subject.

22. The method of solution 20 or 21, wherein a presence of less than 10particles of the pathogen in the biological sample is detected.

23. A method of environmental monitoring, comprising collecting a sampleselected from the group consisting of a soil sample, an aerosol sample,an air sample, or a water sample, contacting the sample with thebiosensor device of any one of solutions 1 to 19, and detecting thepresence of the one or more pathogens in the at least one sample.

24. The method of solution 23, wherein the one or more pathogenscomprise one or more of a heavy metal, a small-molecule agriculturaltoxin, a water-borne bacterial pathogen, an aquatic toxin, a pesticide,an industrial byproduct, an antibiotics, or a pharmaceutical.

REFERENCES

-   [1] S. Khan, A. Ali, H. Shi, R. Siddique, G. Nabi, J. Hu, T.    Wang, M. Dong, W. Zaman, G. Han, Saudi Pharmaceutical Journal 2020,    28, 1004.-   [2] R. de Oliveira Andrade, bmj 2020, 370.-   [3] M. Sorbello, K. El-Boghdadly, I. Di Giacinto, R. Cataldo, C.    Esposito, S. Falcetta, G. Merli, G. Cortese, R. Corso, F. Bressan,    Anaesthesia 2020, 75, 724.-   [4] Y. Song, J. Song, X. Wei, M. Huang, M. Sun, L. Zhu, B. Lin, H.    Shen, Z. Zhu, C. Yang, Analytical Chemistry 2020, 92, 9895.-   [5] L. Zhang, X. Fang, X. Liu, H. Ou, H. Zhang, J. Wang, Q. Li, H.    Cheng, W. Zhang, Z. Luo, Chemical Communications 2020, 56, 10235.-   [6] Z. Daniloski, T. X. Jordan, J. K. Ilmain, X. Guo, G.    Bhabha, B. R. tenOever, N. E. Sanjana, eLife 2021, 10, e65365.-   [7] L. Zhang, C. B. Jackson, H. Mou, A. Ojha, E. S. Rangarajan, T.    Izard, M. Farzan, H. Choe, bioRxiv 2020, 2020.06.12.148726.-   [8] J. A. Plante, Y. Liu, J. Liu, H. Xia, B. A. Johnson, K. G.    Lokugamage, X. Zhang, A. E. Muruato, J. Zou, C. R.    Fontes-Garfias, D. Mirchandani, D. Scharton, J. P. Bilello, Z.    Ku, Z. An, B. Kalveram, A. N. Freiberg, V. D. Menachery, X.    Xie, K. S. Plante, S. C. Weaver, P.-Y. Shi, Nature 2021, 592, 116.-   [9] Z. Ding, X. Liu, X. Ren, Q. Zhang, T. Zhang, Q. Qian, W. Liu, C.    Jiang, Discovery medicine 2016, 21, 331.-   [10] US Food and Drug Administration, US FDA, Silver Spring, Md.    2007.-   [11] N. R. Pollock, T. J. Savage, H. Wardell, R. Lee, A. Mathew, M.    Stengelin, G. B. Sigal, medRxiv 2020, 2020.11.10.20227371.-   [12] Y. Pan, D. Zhang, P. Yang, L. L. M. Poon, Q. Wang, Lancet    Infect Dis 2020, 20, 411.-   [13] A. D. Winder, A. S. Mora, E. Berry, J. R. Lurain, Gynecol Oncol    Rep 2017, 21, 34.-   [14] G. M. S. Ross, D. Filippini, M. W. F. Nielen, G. IJ. Salentijn,    Anal. Chem. 2020, 92, 15587.-   [15] W. F. Garcia-Beltran, E. C. Lam, K. S. Denis, A. D.    Nitido, Z. H. Garcia, B. M. Hauser, J. Feldman, M. N.    Pavlovic, D. J. Gregory, M. C. Poznansky, Cell 2021, 184, 2372.-   [16] U. FDA, n.d.-   [17] S. Guo, K. Liu, J. Zheng, Int J Biol Sci 2021, 17, 1476.-   [18]W. Tai, L. He, X. Zhang, J. Pu, D. Voronin, S. Jiang, Y.    Zhou, L. Du, Cellular & Molecular Immunology 2020, 17, 613.-   [19] M.-Y. Wang, R. Zhao, L.-J. Gao, X.-F. Gao, D.-P. Wang, J.-M.    Cao, Frontiers in Cellular and Infection Microbiology 2020, 10, 724.

CONCLUSION

The above detailed description of embodiments of the technology are notintended to be exhaustive or to limit the technology to the preciseforms disclosed above. Although specific embodiments of, and examplesfor, the technology are described above for illustrative purposes,various equivalent modifications are possible within the scope of thetechnology as those skilled in the relevant art will recognize. Forexample, although steps are presented in a given order, alternativeembodiments may perform steps in a different order. The variousembodiments described herein may also be combined to provide furtherembodiments.

From the foregoing, it will be appreciated that specific embodiments ofthe technology have been described herein for purposes of illustration,but well-known components and functions have not been shown or describedin detail to avoid unnecessarily obscuring the description of theembodiments of the technology. Where the context permits, singular orplural terms may also include the plural or singular term, respectively.Further, while advantages associated with some embodiments of thetechnology have been described in the context of those embodiments,other embodiments may also exhibit such advantages, and not allembodiments need necessarily exhibit such advantages to fall within thescope of the technology. Accordingly, the disclosure and associatedtechnology can encompass other embodiments not expressly shown ordescribed herein.

What is claimed is:
 1. A biosensor device for detecting one or morepathogens, comprising: a detection chip, comprising: a substrate with agraphene surface; a conducting material at a first end and a second endof the graphene surface that form a first electrode and a secondelectrode, respectively; and an insulating material to insulate thefirst electrode and the second electrode, wherein one or more probes areattached to the graphene surface, wherein the one or more probesspecifically bind to one or more target molecules of the one or morepathogens, and wherein the insulating material forms a well to receive abiological sample such that the biological sample is in contact with theone or more probes.
 2. The biosensor of claim 1, wherein at least one ofthe one or more probes is an aptam er.
 3. The biosensor of claim 2,wherein the aptamer comprises a nucleic acid or a peptide, and whereinthe nucleic acid is double-stranded or single-stranded.
 4. The biosensorof claim 1, wherein the one or more pathogens are one or more variantsof a coronavirus.
 5. The biosensor of claim 4, wherein the one or morevariants of the coronavirus include SARS-CoV, SARS-CoV-2, and MERS-CoV.6. The biosensor of claim 1, wherein the target molecule is a nucleicacid or a protein.
 7. The biosensor of claim 1, wherein the targetmolecule includes an S protein of SARS-CoV-2, an N protein ofSARS-CoV-2, a variant thereof, or a subunit thereof.
 8. The biosensor ofclaim 1, wherein the one or more probes comprise multiple probes thatare attached to different portions of the graphene surface.
 9. Thebiosensor of claim 8, wherein a first probe of the multiple probes isattached to a first portion of the graphene surface, and wherein asecond probe of the multiple probes is attached to a second portion ofthe graphene surface that is non-overlapping with the first portion. 10.The biosensor of claim 8, wherein the multiple probes specifically bindto different target molecules of a same pathogen.
 11. The biosensor ofclaim 8, wherein the multiple probes specifically bind to differenttarget molecules of different pathogens.
 12. The biosensor of claim 8,comprising: a plurality of detection chips comprising the detectionchip, wherein each of the multiple probes is attached to a correspondingdetection chip of the plurality of detection chips.
 13. The biosensor ofclaim 1, wherein a handheld device is configured to receive thebiosensor device, and wherein the handheld device is configured toperform a detection of the one or more pathogens based on the one ormore probes specifically binding to the one or more target molecules ofthe one or more pathogens.
 14. The biosensor of claim 13, wherein thehandheld device comprises a wireless transceiver that is configured totransm it a result of the detection, and wherein the wirelesstransceiver supports at least one of a Bluetooth protocol, a Wi-Fiprotocol, or a cellular protocol.
 15. The biosensor of claim 13, whereinthe handheld device comprises: a power source; one or more visualindicators, coupled to the power source, configured to indicate a startand a completion of the detection of the one or more pathogens; and adisplay, coupled to the power source, to present a result of thedetection for each of the one or more pathogens.
 16. A method ofdetecting a presence of one or more pathogens in a biological sample,comprising: receiving, from a subject, the biological sample; contactingthe biological sample with a biosensor device; and determining, based onthe contacting, whether the one or more pathogens are present in thebiological sample, wherein the biosensor device comprises: a detectionchip, comprising: a substrate with a graphene surface; a conductingmaterial at a first end and a second end of the graphene surface thatform a first electrode and a second electrode, respectively; and aninsulating material to insulate the first electrode and the secondelectrode, wherein one or more probes are attached to the graphenesurface, wherein the one or more probes specifically bind to one or moretarget molecules of the one or more pathogens, and wherein theinsulating material forms a well to receive the biological sample suchthat the biological sample is in contact with the one or more probes.17. The method of claim 16, wherein the biological sample is selectedfrom the group consisting of saliva, exhaled breath, nasal swab, ornasopharyngeal swab of the subject.
 18. The method of claim 16, whereina presence of less than 10 particles of the pathogen in the biologicalsample is detected.
 19. A method of environmental monitoring,comprising: collecting a sample selected from the group consisting of asoil sample, an aerosol sample, an air sample, or a water sample;contacting the sample with a biosensor device; and detecting thepresence of the one or more pathogens in the sample, wherein thebiosensor device comprises: a detection chip, comprising: a substratewith a graphene surface; a conducting material at a first end and asecond end of the graphene surface that form a first electrode and asecond electrode, respectively; and an insulating material to insulatethe first electrode and the second electrode, wherein one or more probesare attached to the graphene surface, wherein the one or more probesspecifically bind to one or more target molecules of the one or morepathogens, and wherein the insulating material forms a well to receivethe sample such that the sample is in contact with the one or moreprobes.
 20. The method of claim 19, wherein the one or more pathogenscomprise one or more of a heavy metal, a small-molecule agriculturaltoxin, a water-borne bacterial pathogen, an aquatic toxin, a pesticide,an industrial byproduct, an antibiotics, or a pharmaceutical.